Hee-Jin,
My understanding is that the fluorescence of typical GFP is
pH sensitive being quenched effectively at acid pH. The plant
apoplast is typically pH 5.5 more or less. So it is possible that you
are not seeing the cell wall GFP for that reason. You could try
perfusing your sample with neutral buffer (although it can be tough
to reach the microdomains deep in the apoplast) or fixing and
staining with an antiGFP antibody (this may be more trouble than you
want).
Hope this helps,
Tobias
>Dear Arab-gen and colleagues
>>How are you doing?
>>Does anybody know if overly expressed GFP with 35S can be found
>extracellular fraction of cells in addition to the typical
>localizations (cytoplasm and nucleoplasm) of GFP ? One of my
>soluble protein (no signal peptide), abundantly over-expressed
>mGFP5 fusion with 35S was found in the cell wall fractions by
>Western blot although microscopic picture showed most portion of the
>fusion protein is in cytoplasm.
>>I found a paper that 31% of overly expressed mGFP5-ER with 35S in
>BY2 was secreted into medium according to Western blot analyses (Liu
>et al., 2001, J Biotechnology 87: 1-16). However, I could not find
>so far if overly expressed GFP alone in plant cells can be
>secreted.
>Any comment how the levels of GFP expressions can affect
>localization would be appreciated.
>>Hee-Jin
>>>>>>_______________________________________________
>Arab-gen mailing list
>Arab-gen At net.bio.net>http://www.bio.net/biomail/listinfo/arab-gen
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